BeQuanti: software for automatic
detection and analysis of cell
adhesion underflow
Lara Toffali1, Michela Farenzena2, Roberto Marzotto2 and Carlo Laudanna1
1
Laboratory of Cell Trafficking and Signal Transduction
Department of Pathology and Diagnostic,
Division of General Pathology, University of Verona
2
eVS embedded Vision Systems, Verona
Immune system: right site at right moment
The multistep model of leukocyte recruitment
Blood flow
Tethering and Rolling
Activation
Arrest
Mucins, selectins, integrins
Chemokines or Chemoattractans
Integrins
Stabilization
Spreading
Transmigration
Endothelial cells
ms
Immunodeficiency, autoimmune diseases and cancer
s  min
Bioflux facility in Laudanna’s Laboratory
Laboratory of Cell Trafficking and Signal Transduction
Department of Pathology and Diagnostic, Division of General Pathology
University of Verona
Bioflux facility in Laudanna’s Laboratory
Glass capillaries vs Bioflux
microfluidics
Glass capillary
Bioflux microfluidics
Underflow adhesion assays with Bioflux
Leukocytes adhesion uderflow at
2 dyne/cm2
Selectin
ICAM-1
Chemokines
Recording: 25 frames/s
Movie length: 5-10 minutes
=
Total frames: 7500-15000
Manually performed
movie analysis
Limited
Time consuming
Prone to errors
Tedious
BeQuanti
(for Behavior Quantification)
The Automated Tool for Cell Behavior Quantification
Laboratory of Cell Trafficking
and Signal Transduction
Carlo Laudanna
Roberto Marzotto
Lara Toffali
Michela Farenzena
BeQuanti comparison with other packages
Leucotrak: Willenbrock, F. et al., Biophys J. 105, 1110-22 (2013).
ImageJ: Meyer dos Santos, S. et al. Platelets. 21, 60-6 (2010).
ImagoQuant: Giegold, O. et al., Exp Dermatol. 18, 238-45 (2009).
BeQuanti: licensing
» Trial free license
Feature
Limitation
Time period
30 days
Number of sequences per video
Max 2 sequences
Sequence length
Max 500 frames
Batch mode
Not included
Export results
Not included
» Full license
» No features limitations
» Node lock license
» Updates included up to the next major release
» Unlimited technical support
eVS embedded Vision Systems Srl
www.embeddedvisionsystems.it
info@evsys.it
BeQuanti
The Automated Tool for Cell Behavior Quantification
DV, MPEG,
Mov and AVI
For each frame a list of cell
positions (x, y)
A list of cell, for each cell the frames
and position in which it appears
Tethering, Rolling, Subrolling
Arrest and Unclassified
Round, Polarized,
Spread and Unknown
BeQuanti
The Automated Tool for Cell Behavior Quantification
1. New project
2. Analysis
3. Data
1.
New project and Movie import
1.
Sequence Selection
1.
Edit Analysis Parameters
1.
Adhesion parameters
Every single cell detected
Adhesive behaviors
(events) analysis
Subrolling corresponds to
very short cell movements
detected between two
distinct arrest events for a
defined cell (adhesion
stabilization)
Subrolling threshold:
Spatial Arrest Threshold
S.A.T.
maximal tolerated small cell
movements distinguishing
arrest versus rolling
Temporal Arrest Threshold
T.A.T.
temporal cutoff defining arrest
versus rolling
maximal tolerated small cell
movements (between two
distinct arrest events)
distinguishing subrolling
versus rolling
Cell arrest is fully defined by the combination of S.A.T. and T.A.T.
1.
Adhesion parameters
1.
Global Analysis vs Parametric Analysis
Incremental T. A. T.
0,04 s
3s
Frequency of 1/fps
Fixed T. A. T.
1.
Start analysis
BeQuanti supports
multiprocessor and
multi-core
architectures
BeQuanti
The Automated Tool for Cell Behavior Quantification
2. Analysis
2.
Play Mode: detections
2.
Play Mode: Tracking
2.
Play Mode: Events
2.
Play Mode: Arrested Types
BeQuanti
The Automated Tool for Cell Behavior Quantification
3. Data
3.
Results report: Parametric Analysis
Total
3.
Results report: Parametric Analysis
Normalized
3.
Target an individual cell
directly on the movie
3.
Target an individual cell
directly on the movie
25
3.
Results report: Cell Morphology Analysis
3.
Results report: Cell Morphology Analysis
3.
Results report: Global Adhesion Analysis
0,04 s
3s
Incremental T. A. T.
3.
Results report: Global Adhesion Analysis
0,04 s
3s
Total
3.
Results report: Global Adhesion Analysis
0,04 s
Normalized
3s
3.
Export results (cvs format)
3.
Example: h-PMN with PTx
CELL TYPE:
Human primary neutrophils isolated from healthy volunteers by standard gradient
centrifugation (Ficoll). Contaminating erythrocytes were removed by dextran-500 sedimentation
followed by hypotonic lysis.
TREATMENT:
Primary neutrophils were treated or not with 2 μg/ml Pertussis Toxin (PTx) for 3 h at 37°C
COATING:
1 ug/ml P-Selectin, 5 μg/ml ICAM-1 and 25 nM FMLP
PARAMETERS:
S.A.T. 5 μm – T.A.T. 2s – Sub-rolling threshold 20 μm
PMN treated with Pertussis Toxin (2 μg/ml)
PMN control
0,4
0,21
0,08
0,02
Future developments
Detection of different kind of cells
Analysis of cell behavior on different backgrounds, including adhesion:
- on endothelial cells
- in different experimental settings (Intravital microscopy or glass capillary)
Detection of cell motility underflow, such as cell crawling and transmigration
BeQuanti was developed and tested to analyze underflow experiments performed with BioFlux system
at 20x phase contrast magnification
BeQuanti is fully customizable upon request!
Laboratory of Cell Trafficking and Signal Transduction
Department of Pathology and Diagnostic,
Division of General Pathology
University of Verona
Carlo Laudanna
Roberto Marzotto
carlo.laudanna@univr.it
roberto.marzotto@evsys.it
+39 0458027689
+39 045 8949180
Lara Toffali
Michela Farenzena
lara.toffali@univr.it
michela.farenzena@evsys.it
+39 0458027145
+39 045 8949180
http://dp.univr.it/~laudanna/LCTST/index.html
www.embeddedvisionsystems.it
info@evsys.it
Image processing details
» Cells Detection: cells classification by a
discriminative classifier trained on cells
images, based on gradient orientation
» Cells Tracking: global optimal matching
» Events Classification: exhaustive recursive
search
» Cells Morphology Classification: shape and
color based classification